The formation of tight junction (TJ) barriers between renal tubular cells is an absolute requirement for tubular transport and proper solute, acid-base and water balance. TJ remodeling must also occur during tubulogenesis, tubular repair and all forms of mesenchymal-to-epithelial transformation. Unfortunately, we still know very little about the molecular events that link initial cell-cell contact to assembly of the barrier. Lacking this knowledge, we will not understand how barrier assembly is regulated and in the long term will be unable to manipulate assembly to maintain or induce repair of the tubular barrier. The current proposal is based on recent breakthroughs which convincingly show that the multi-domain scaffolding proteins ZO-1 and ZO-2 are necessary for TJ assembly and are directly involved in linking early spot-like cadherin contacts to continuous adherens junctions and subsequent recruitment of TJ proteins into barrier strands. The goal of this project is to understand how the ZO proteins regulate the interactions between TJ proteins that mediate different steps of TJ assembly. Studies will be conducted in renal cultured cell models. Aim 1 will test the hypothesis that binding of ZO-proteins to the transmembrane proteins occludin and tricellulin is required for TJ strand assembly, and assembly is regulated by the Unique-6 domain of ZO proteins. We will test how ZO-1, occludin and tricellulin are required for renal epithelial cells to form 3D cysts in culture. siRNA silencing and expression of mutated proteins in cultured renal epithelial MDCK cells will provide the major technical approach. Aim 2 will test the hypothesis that ZO-proteins promote E cadherin-mediated adhesive complexes by promoting cell-cell adhesion and/or adherens junction assembly. We will use RNAi silencing and transgene rescue to identify the molecular interactions with promote ZO-1 activity at the adhesive contacts that are required for TJ assembly. Aim 3 will test the hypothesis that ZO-1 promotes the de novo assembly and/or recruitment of f-actin at cell-cell contacts. Aim 4 will use x-ray crystallography to elucidate the structural basis for the interaction between ZO-proteins and occludin/tricellulin and their regulation by the calcium-sensor calmodulin and the Unique-6 domain. We are in an ideal position to achieve these aims because of our past experience and contributions to the field, preliminary studies demonstrating feasibility and appropriateness of our models, availability of reagents and a history of synergistic collaboration between our cell biology (UNC) and structural (UIC) teams. The significance of these results is that they will define basic cellular mechanisms required for TJ assembly, which is fundamental to normal kidney function and altered in disease. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding at a molecular level how the renal epithelial cell tight junction barrier is formed. The public health significance is that loss of the barrier leads to acute renal failure such as is common following ischemic and toxic injury. The findings will guide strategies to preserve and restore the tubular barrier when injured.